CN112616858B - Application of LOC663462 gene in pest control - Google Patents

Application of LOC663462 gene in pest control Download PDF

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CN112616858B
CN112616858B CN202011504737.9A CN202011504737A CN112616858B CN 112616858 B CN112616858 B CN 112616858B CN 202011504737 A CN202011504737 A CN 202011504737A CN 112616858 B CN112616858 B CN 112616858B
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朱子丹
杨宏广
童春梅
邓惠敏
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Abstract

The invention relates to the technical field of genetic engineering, in particular to application of LOC663462 gene in pest control. According to the invention, the LOC663462 gene is used as a target, dsRNA is designed and injected into an insect body, the influence on the reproduction and development of the insect is observed, and the insect body type is reduced, and the body length and the body width are reduced to a certain extent after the LOC663462dsRNA is injected; the egg laying number and the hatching rate are obviously reduced after selfing, and the LOC663462 gene influences VgR to recognize vitellogenin by influencing the expression of a vitellogenin receptor VgR, so that the vitellogenin is influenced to enter the ovum to participate in the maturation process of the ovum, and the LOC663462 gene plays an important role in influencing insect reproduction, particularly pest reproduction.

Description

Application of LOC663462 gene in pest control
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of LOC663462 gene in pest control.
Background
Tribolium castaneum (Tribolium castaneum) belongs to the family Bacteroides Coleoptera, and Triboehira, and is distributed in tropical and warmer areas of the world. The tribolium castaneum has complex feeding property, mostly lives in dry environment, has the characteristics of long service life, quick propagation, wide adaptability and the like, and is an important worldwide pest for grain storage. Besides direct damage, the stinky glands on the body surface of the adult tribolium castaneum can also secrete stink liquid containing carcinogenic substance benzoquinone, so that the damaged substances generate fishy smell and moldy smell, and the damaged substances are discolored and smelly and cannot be eaten.
The tribolium castaneum is a representative insect which is recognized as an insect of coleoptera and is easy to be experimentally manipulated, and is also a model insect which is easy to be manipulated in the aspects of genetic evolution and developmental research. The complete genome sequencing of the mutant shows that the genome of the Tripsammosile castanea comprises about 2 hundred million nucleotides, and the number of genes (or proteins) is about 16000. The comprehensive analysis of the genome can help to discover new drugs and antibiotics, help to control agricultural insects and germs for resistant species, pesticide effect and biological agents, and finally bring great promoting effects to the development of various disciplines such as insect molecular ecology, insect taxonomy, insect toxicology and the like.
RNAi refers to target gene homologous double-stranded RNA (dsRNA) causing the degradation of mRNA encoded by the gene, thereby specifically inhibiting the expression of the gene, and the RNAi has the characteristics of high efficiency and specificity and is widely existed in various organisms such as animals, plants and the like. The tribolium castaneum is an insect with a high-efficiency RNAi mechanism, namely dsRNA is injected into any period of larvae, pupae, adults and embryos of the tribolium castaneum, so that the expression of a target gene can be inhibited, and the inhibition effect can be continued to the next generation. Therefore, the method screens out key targets influencing the development, survival and the like of the tribolium castaneum, and has important significance for accelerating the RNAi technology in the aspect of pest control.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. The inventor designs dsRNA by taking LOC663462 gene as a target, injects the dsRNA into the tribolium castaneum body, and finds that the development of ovaries of the tribolium castaneum is obviously inhibited, the egg laying number and the hatching rate are obviously reduced, and the worm bodies of the hatched adults are obviously smaller. The LOC663462 is shown to be an important target gene affecting the tribolium castaneum.
The technical scheme of the invention is shown as follows.
The invention provides an application of LOC663462 on one hand, wherein the nucleotide sequence of the LOC663462 gene is shown in SEQ ID NO. 1; the application comprises the following steps: regulating diapause of insects, preparing products for regulating diapause of insects, preventing and controlling pests or preparing products for preventing and controlling pests.
According to some embodiments of the invention, the pest is a coleopteran insect, preferably a tribolium castaneum.
According to some embodiments of the invention, the insect is a coleopteran insect, preferably a tribolium castaneum.
The invention also provides application of the LOC663462 gene as an inhibition target or a silencing target for preparing an interference molecule for specifically interfering the expression of the harmful insect gene or inhibiting the growth of the harmful insect, wherein the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1.
According to some embodiments of the invention, the interfering molecule is a construct of the LOC663462 gene or its transcript as a dsRNA, antisense nucleic acid, small interfering RNA, microrna that inhibits or silences a target, or a construct capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microrna.
According to some embodiments of the invention, the pest insect is a coleopteran insect, preferably a tribolium castaneum.
In another aspect of the present invention, there is also provided a method for controlling harmful insects, the method comprising: down-regulating the expression or activity of LOC663462 gene in the body of the harmful insect; wherein, the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1.
According to some embodiments of the invention, the method of down-regulating the expression or activity of the LOC663462 gene in a pest insect comprises: knocking out or silencing LOC663462 gene in genome of harmful insect; or transferring a down regulator for down-regulating LOC663462 gene transcription, polypeptide expression or polypeptide activity into a harmful insect body.
According to some embodiments of the invention, the pest insect is a coleopteran insect, preferably a tribolium castaneum.
The invention also provides dsRNA for inhibiting the expression of the LOC663462 gene, wherein the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1.
According to some embodiments of the invention, the primer sequences for synthesizing the dsRNA are shown as SEQ ID NO 2 and SEQ ID NO 3.
In yet another aspect, the invention provides a biological product comprising a dsRNA as described above.
According to some embodiments of the invention, the biological product is a spray formulation or a bait.
The invention has the beneficial effects that:
the invention takes LOC663462 gene as a target, designs dsRNA and injects the dsRNA into an insect body, observes the influence on the reproduction and development of the insect, and discovers that after the LOC663462dsRNA is injected, the body size of the insect is reduced, and the body length and the body width are reduced to a certain degree; the egg laying number and the hatching rate are obviously reduced after selfing, and the LOC663462 gene influences VgR to recognize vitellogenin by influencing the expression of a vitellogenin receptor VgR, so that the vitellogenin is influenced to enter the ovum to participate in the maturation process of the ovum, and the LOC663462 gene plays an important role in influencing insect reproduction, particularly pest reproduction.
Drawings
FIG. 1 is a diagram showing the expression of LOC663462 gene in different periods of Trimerella castanea;
FIG. 2 is an electrophoretogram of synthetic dsRNA;
FIG. 3 is a diagram showing the expression of LOC663462 gene at different times after dsRNA injection;
FIG. 4 is a phenotype diagram of the adult tribolium castaneum 1h after dsRNA injection;
FIG. 5 is a phenotype plot of adult Tripsammophila 1 day after dsRNA injection;
FIG. 6 is a graph showing the statistical results of the length and width of the adult Tripsammophila after dsRNA injection;
FIG. 7 is a statistical graph of the number of eggs laid by the tribolium castaneum and the hatching rate after dsRNA injection;
FIG. 8 is a graph of the change in Tripsammosile castanea ovaries after dsRNA injection;
FIG. 9 is a graph of another variation of Tripsammosile castanea following dsRNA injection;
FIG. 10 is a view of an ovum after dsRNA injection;
FIG. 11 is a view of a single ovum after dsRNA injection;
FIG. 12 is a graph of vitellogenin content in adult hemolymph and ovaries following dsRNA injection;
FIG. 13 is a graph showing the change of expression levels of LOC663462 and vitellogenin receptor in the ovaries of adults after dsRNA injection.
Detailed Description
The technical solutions and effects of the present invention will be further described and illustrated with reference to the following specific examples, but the present invention is not limited to these specific embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
Insects: derived from the tribolium castaneum raised by the institute of insect science and technology of university of Master in south China.
Reagent: RNA extraction Reagent TRIzol Reagent (ambion by life technologies, product number 230011), gene expression quantification Reagent iTaq Universal SYBR Green Supermix (BIO-RAD, product number 1725121), Reverse Transcription Kit High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (applied biosystems by Thermo Fisher Scientific, product number 00978080), dsRNA Synthesis Kit HiScriber T7 Quick High Yield RNA Synthesis Kit E2050S (New England Biolabs, product number 10056893).
In the experiment, each group of 30 head worms is observed and counted in each batch of experiments. The RNA-extracted material was 3 replicates per time point for each batch of experiments, and 3 worms were collected as one sample.
Example 1 expression of LOC663462 Gene in different periods of Trimerella castanea
The expression condition of the LOC663462 gene is detected in the Siberian beetle by using quantitative PCR, and the quantitative primers are as follows:
F’：GCAGTTCAAGCGCAAAGACA(SEQ ID NO:4)，
R’：TTTCACCGGTTGAGAGACGG(SEQ ID NO:5)；
the internal reference gene is RP3, and the primer sequence is as follows:
F’：TCAAATTGATCGGAGGTTTG(SEQ ID NO:6)，
R’：GTCCCACGGCAACATAATCT(SEQ ID NO:7)；
the detection period comprises the 1 st day (EggD1) of the egg stage, the 4 th day (EggD4) of the egg stage, the 18 th to 20 th days (L-D18-20) of the larva stage, the 1 st to 5 th days (P1-5) of the pupal stage, 0.5-1 hour (Ad 0.5-1 h) after eclosion of imagoes and 1-6 days (Ad 1-6) after eclosion of the imagoes.
As shown in figure 1, the expression of LOC663462 gene in different periods of tribolium castaneum, it can be seen that the expression of LOC663462 gene is low in egg stage and larval stage, and has high level expression in the first day of pupal stage, and the expression of LOC663462 gene is maintained at medium level expression in 2-5 days of pupal stage, and its expression is obviously increased in 1 day of adult, and its expression is reduced in 3 days and 6 days.
Example 2 Synthesis of dsRNA of LOC663462 Gene and its interference efficiency
Primers for synthesizing dsRNA were:
F’：TAATACGACTCACTATAGGGATGGATACGATTGTGCAAGG(SEQ ID NO:2)，
R’：TAATACGACTCACTATAGGGCCGTTTCCTGGTCAGTTAT(SEQ ID NO:3)；
FIG. 2 shows an electrophoretogram of synthetic dsRNA; from left to right, the first lane is marker, the second lane is DNA template for synthesizing dsRNA, and the third lane is synthesized dsRNA, wherein the dsRNA is 500 bp.
With GFP dsRNA as a control, primers for synthesizing GFP dsRNA were:
F’：TAATACGACTCACTATAGGGTGGTCCCAATTCTCGTGGAAC(SEQ ID NO:8)，
R’：TAATACGACTCACTATAGGGCTTGAAGTTGACCTTGATGCC(SEQ ID NO:9)；
and selecting to carry out dsRNA injection on the next day of pupal stage according to the expression conditions of the LOC663462 gene in different periods of the tribolium castaneum. And respectively injecting 200ng dsRNA into each head of the tribolium castaneum pupae, and taking materials at 0h, 48h, 72h, 96h and 120h after injection to detect the expression quantity of the LOC663462 gene so as to detect the RNA interference efficiency.
As shown in fig. 3, which is a graph showing the expression of the LOC663462 gene at different times after dsRNA injection, it can be seen that the expression level of the LOC663462 gene is significantly reduced after 96 hours after the injection of the LOC663462dsRNA compared with the GFP dsRNA injection control.
Example 3 Effect of the LOC663462 Gene dsRNA on the Tocopherol phenotype
Pupae emerged into adults 96 hours after dsRNA injection.
Adults 1h after eclosion were observed, and as shown in fig. 4, no significant difference in appearance from the control group was found.
Adult insects 1 day after eclosion were observed, and as shown in fig. 5, it was found that the bodies injected with the LOC663462dsRNA were also not significantly different in appearance from the control group, but slightly reduced in size from the naked eye, regardless of female or male.
The body length and the body width of the adult after 1 day of eclosion are counted, and the results are shown in fig. 6, and it can be seen that in the male worms, the body types of the Wild Type (WT) and the GFP dsRNA (dsGFP) injected are closer, and the body length and the body width of the adult with LOC663462dsRNA (dsLOC663462) injected are both smaller than the body of the adult with GFP dsRNA (dsGFP) injected, and have statistical difference; in female insects, the length and width of Wild Type (WT), adult injected with GFP dsRNA (dsGFP) and adult injected with LOC663462dsRNA (dsLOC663462) are reduced in sequence, but no statistical difference exists.
Example 4 Effect of LOC663462 Gene dsRNA on Trimeresurus immaturus reproduction conditions
1. Influence of LOC663462 gene dsRNA on number of eggs laid by tribolium castaneum and hatchability
The results of observing and counting the number of eggs laid and the hatchability after the selfing of the tribolium castaneum are shown in fig. 7, and it can be seen that the number of eggs laid and the hatchability of the worm injected with LOC663462dsRNA (dsLOC663462) are both obviously reduced. The average egg laying rate per insect per day of the control group (dsGFP) is 8, the average egg laying rate per insect per day of the control group injected with LOC663462dsRNA is 1, the egg laying rate is reduced by about 87.5 percent compared with that of the control group, the average hatching rate of the control group is 82 percent, the average hatching rate of the control group injected with LOC663462dsRNA is 6 percent, and the hatching rate is also reduced by about 92 percent compared with that of the control group.
2. Influence of LOC663462 gene dsRNA on red Valley ovary
The 7 th day ovaries of the adult tribolium castaneum are dissected and observed, and the results are shown in fig. 8 and 9, it can be seen that the ovaries of the control group (dsGFP) are good in development form, ova in different development stages can be seen in the oviduct, while ovaries of the ovaries injected with LOC663462dsRNA (ds LOC663462) are in two forms, one form is that the ovaries contain a large amount of ova in a later development stage, but the shape of part of the ova is irregular (fig. 8), the other form is that the ovaries are obviously smaller, only a small amount of ova are contained in the ova in the development stage, and the ovaries with one side are slow in development (fig. 9), and female insects with two types of ova can only produce a small amount of ova.
3. Influence of LOC663462 gene dsRNA on number of eggs laid by tribolium castaneum and hatchability
Mature ova in the ovaries of adult tribolium castaneum 7 days are taken out and observed, as shown in fig. 10, the ova in the control group (dsGFP) are good in development form, oblong, glossy in surface and uniform in egg color, while the ova in the ovaries of adult larvae injected with LOC663462dsRNA (dsLOC663462) are irregular in appearance, slightly shorter and more round than the control group, the egg shells are partially transparent, and the colors in the ovaries are not uniform.
Further observation of individual eggs revealed that regular lines were visible on the surface of the control (dsGFP) eggs, whereas no lines were visible on the surface of the LOC663462dsRNA injected (dsLOC663462), the egg shell was partially transparent and the distribution of the egg contents was not uniform, as shown in figure 11.
4. Influence of LOC663462 gene dsRNA on Trimeresurus terrestris vitellogenin
From the above results, it was analyzed that egg yolk protein accumulation of eggs in adults injected with LOC663462dsRNA (dsLOC663462) should be less than that of control group (dsGFP), since the ovary of the adult beetle starts to develop at day 1 until day 5 of ovarian maturation. The vitellogenin is synthesized by fat bodies, then secreted into haemolymph, transported into ovaries by haemolymph, recognized by vitellogenin receptors and absorbed into the ovaries to participate in the maturation process of the ova.
To investigate whether LOC663462 affects the vitellogenin synthesis process, hemolymph of a control group and an adult LOC663462dsRNA injected are respectively taken and subjected to SDS-PAGE analysis, and the results are shown in FIG. 12, which shows that the content of the vitellogenin in the hemolymph of the adult LOC663462dsRNA injected is more than that of the control group (FIG. 12A), while the content of the vitellogenin in ovaries of the adult LOC663462dsRNA injected for 5 days and 7 days is obviously lower than that of the control group (FIG. 12B), indicating that the content of the vitellogenin in the ovaries is not affected after the injection of the LOC663462dsRNA, and the content of the vitellogenin is affected, so that the vitellogenin is accumulated in the hemolymph.
5. Mechanism of LOC663462 gene affecting tribolium castaneum vitellogenin
According to the above results, it is presumed that LOC663462 is likely to affect the accumulation of vitellogenin of the egg to affect the development of the egg, and in order to further study the mechanism that LOC663462 affects the vitellogenin entering the egg, the expression levels of LOC663462 and vitellogenin receptor (VgR) in the ovary of adult worm after dsRNA injection are respectively detected, and as a result, as shown in fig. 13, it is found that the expression levels of LOC663462 (fig. 13A) and VgR (fig. 13B) are significantly lower than those of the control group in the ovary of adult worm injected with LOC663462dsRNA, indicating that LOC663462 affects VgR to recognize vitellogenin by affecting the expression of the vitellogenin receptor VgR, thereby affecting the vitellogenin to enter the egg to participate in the maturation process of the egg, which has an important role in affecting insect reproduction, especially reproductive pests.
While the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the following claims be interpreted to include all such embodiments and equivalent variations. In addition, the contents of all references cited herein are hereby incorporated by reference.
SEQUENCE LISTING
<110> university of south China
Application of LOC663462 gene <120> in pest control
<130> 111
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 6595
<212> DNA
<213> Tribolium castaneum
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tatttccgtt gagtgacgcg agtggaattt acatatttta ttatctcttc ggcatgatgt 1560
aatttcgata gaatattacg ggcgtgataa caaataagtc aaaaacgtca atgttttaag 1620
gtgttttgtt tttaatttta ttacgcgctg ttatgtagtt taattcaaac aaaaacacaa 1680
acttctaata attattcaaa acaaatcgca acaaatcaca caatatatta aaattttact 1740
gtactaagat gtttaaactg cagtccgtgt ccaatttttt tgttttgttt ggatttttta 1800
aattgtttat tattccggaa tcaacaaaaa tgttgttact gaaaactcaa caaattgtcg 1860
aatttgttgg ttatttgctg tttatttatt attattttat tatgcaatcg tgattaaaga 1920
ttcttttttt tattgtaaaa aataataaaa aagcacccga aataaacgca aaaattacga 1980
aaaatatcaa gattttcttg taaatattaa taattaaata aaggaaactg aacaaaatgt 2040
catgtctttt tttaaagaat tttttagttg ggaaaataaa ttcatttaaa ctcttaactg 2100
ttcttaaaag aagaatttta gaattttggc gaaaaatagt ttcccaaaaa gacgttcaag 2160
ctgcggtctg cgttgcataa tttttttttt tttttttgaa aaatttaaag aagccacgaa 2220
aatatcaatt tttttcagaa atgatatttt ccaaacatgt taagactacg gttcgccgcg 2280
cattttcttt ttttttaact acatattagc ttagaatcag gaaaaaagtg ttaattcagg 2340
caaaaacttt ccaaaataat taagaatgtt gaagaaactc taacaaaata taacggaatt 2400
cggaatttct tcagttgaga aaataaaaaa gttaaacatt tgcacaaaaa aataccaaca 2460
ttttgtcgga aaagcaattt tccaaaaaga tgttttgact gcggtccgta tcgcaatttt 2520
ttaaatcaca attagaggaa aaaagtgtta ctaaaggcaa aaacatgcta aaataattaa 2580
aaatgttaaa gaaacctgaa caaaatatga agaaaaattc agaattcaga gaaaataaaa 2640
aacaccaaat aaccaaaatt ttgttggaaa aatattttac gactaaaatg tttagtctgc 2700
ggtcggcttc acattttttt attgtggtaa aatcaggaaa agagacaaaa aaacatgcca 2760
aaatgaaaaa acttgaacaa aatgttacgg aaattcagat tttttcagtt gggtcaaaca 2820
cttgcacgag aaataggtgt ttagtctgcg gtcgacctca cattttttta aattctaatc 2880
aaaaaaaaaa aaacaaaaaa ggaaacttgg acaaaatatc gaatttggca ctcacttgct 2940
tttatggtcg cgaaaagttg ttctcactcc catgcccgac ctggcaaaac aaatacccgc 3000
cgccgcccgc tttgattaaa tacttgagct agtcacgtga ctcctccacc taagtgactt 3060
ggtattcgtt ccagcactgg gcgtttttgc caagaaaaca atccctcgaa gcacacagtt 3120
cggacctttg actggaattc tgaaaccgag caccgaacgt ttgaaaaacg gtccgacgtc 3180
tttgcaattt tttatcgaag atgagaacaa cgtcgtgtca cagattgatg tttcggatga 3240
aagtaagtcg aattgtgagt ttatagtttc gaaaaaagtc gtgtttagat gcttcgaatt 3300
ggatgcgttt tatcagacaa gcgacgaatt ataacgagca aaatttattg ataacgcagg 3360
aaggcaactc tttgtatttc acaacgacga ggagcattct tccgaagcag gaactgaagg 3420
taggtgtatg cttgtgtggc gtttttgcct aatttttggg ttcgtaaggt tggctacagt 3480
gtgccctacg ccacccgtca caaattatca attctggtcc ccgaggaaga aaaatcgtgg 3540
ccttgttacg aatgttcgga aacttttgcc tcctctgatg aattacagaa acatttgaac 3600
atccacgaca gtgacgagga aaacaaatgg aagaaaaaga atttaaaaaa taagaaaaaa 3660
atcccaaaaa gtgagacgtt tcaatgtaat aattgcaacg aactgttcgt ccagccggga 3720
aaagtcgccc tcaggcaaca cttgattgaa aaacaccttt tgagcggttt ggacttgatc 3780
gatcagtatt tttcttcagt catgtgagtg tcttaagcga aaataattcc cccgaaacca 3840
cgtttttcca caggaattac aagtgcgata aatgcgaatt ggttttcaat tcggaacctt 3900
tgttaaaaat ccataactat cttcatgatt cggacagttc ggacgaacaa acgaatcatg 3960
tttgtcccaa ctgtcagaaa aaattcccaa cgcaacgcca acttgttacg cacgtcgcta 4020
cgcacgcact taccaaaaaa ccggaaacgg aaacgttcaa gtgtccagtg tgccacaaaa 4080
tgttcgctat gcgcgagcga ctaagggtta gtgtgggttt tgacatgttt atttccacgc 4140
gaaattcgca aataatttca aattttcacg cgatttagcc aacttcgcag tcttcaattt 4200
ccattgatta aaaatggcaa aagtaacatc attttcagtt taattcggtc gtttttagca 4260
ttttttgtca aaatttcgcc aaatcaaaaa acatatttgg aagaaaaaca agataaaaag 4320
ctctgaatag ggcaagaatt gcacaatgtt ttaactaact taatttttag cgaaatttcg 4380
cgctataatt gcatgttttt tgattaaaaa acgaataaga tgtccataaa ttttatccaa 4440
ttgatcaaaa actgcaattg ttactcaaaa atgtgtttca acgcaacgaa aatactcgac 4500
tttagctaat ttgctcgctg tttgcgtccc tttctagttt ttgtatgcgc attttatgta 4560
aaattcttat ttttagtgtc gtcccgtcaa ggtaagtttt tttattttat taggtattta 4620
gtaaacaatt tcggaatatt attacaacaa tcacaagaaa actgagtttt tttaacactc 4680
aaaaaattgt tatgataact ttgatttcat tttaaaaata taaatcaggc aatttgattc 4740
caaatttaat tgttccggac agtaaaataa atatttgtcg gctatgtcta attaataaaa 4800
aaaaaaaaaa acgtttcgcg tggaaataga cacgttattt ttattgaaaa tagagagtga 4860
ctgaagcaat tgcagcgcca catgctggtg cacggctctg acgactcgaa gcctctccag 4920
tgtaaaactt gcaataaacg ctttgtgaac aattcggcgc tcgccggcca catcaagacc 4980
caccttgtgg gcaaaaaaat cttcgaatgt cccatttgta aagaaaattt cgaccacgtc 5040
ctaaaactga aactccacgt ccccaaacac tgcgaaaata acacatactc gtgccctcat 5100
tgcagcaaag ttttcaagaa gtacagtatc atacgcaaac atatccgtgc gtttcactgc 5160
gaccagaaac acgcatgtcc ccactgtatt aaaatgttcc cgactttgga taaattaaaa 5220
atgcatttat tgcgccattc ggaccaccgg gagtttttat gcgccgattg tggcaagcag 5280
ttcaagcgca aagacaagct gaaagagcac tgcaagcgca tgcattcgga ggagcgcgaa 5340
aacgacgttc caaggccccc gaaacccgtc tctcaaccgg tgaaaaaact caatccgaag 5400
agcgaaccga ccgatttcca ccgattcatc tacaagtgtc acacgtgtct tgttgggttt 5460
aaacgtaggg ggatgttggt taaccacttg gcgaagcgac acccggatat aagcccggat 5520
tcggtcccgg aattgaattt accgattttg caaacgacgc gtgactatta ttgccaacac 5580
tgcgataaag tttataagag tagttcgaaa cgcaaagctc acattttgaa gaatcatccg 5640
ggggccgctt tgccgatgag taaccggaaa cagggcaatt ttccggataa ttccgggctt 5700
ccgaatccga cgttttcgca aactgtgggc agtattacga cgcggccgca gaactgcaag 5760
tggtgtcata agcagtatgc gagcaaagcg aagttgttgc agcatcagag gaagaagcac 5820
agcgagcagt tggctcagca gaatgggggc aaggaggagc agaagtacac gccgccggag 5880
caggcggagg gacaacagaa tgacttgcag gactttaagg ttggttttat gccgcaggaa 5940
tcactatgtt tagtccgcgg tccgcgctct catttttttt ttttaataat atagcaagta 6000
ataaaaaaat gttaatgttt ccgcaggtgt ctgagagcga tattttgaac agcgccctcg 6060
aagaatataa caaacgcgga ggggagcagt tttgtcattt tgttgcgatt gaaggtgggg 6120
acgagtttga gcagtcaact ggggatttgg gggcccccaa ttcgcatttg tttcgcttac 6180
tgacaacgag caacggtgaa tattttcccc gagataaaaa aaattgattg tgttttagaa 6240
cttccaggtt tagtgccacc acgttgaaga aaaacctcga ggatttcgag tatttccggt 6300
gctcctttgt gagatttcca ctcaactgag tgattttgag cgagtttgtg ccgtaggttg 6360
aaacctacgt atttaataaa atgcaaaaag taacaaaata gaacgagagg cgaagagaat 6420
tttgggaaat tgagattatt ttagcaataa ttgtcttaat tgtaacaaat ttataaacga 6480
acaagcgctt attgtttggc ttggccgcgg ccgcttgtgt ttagcttagg gacgtttgtt 6540
tgggtcaaaa atgtatccta gattctaaaa gaaatgaaac cttttccccg tgata 6595
<210> 2
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
taatacgact cactataggg atggatacga ttgtgcaagg 40
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
taatacgact cactataggg ccgtttcctg gtcagttat 39
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gcagttcaag cgcaaagaca 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
tttcaccggt tgagagacgg 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
tcaaattgat cggaggtttg 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
gtcccacggc aacataatct 20
<210> 8
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
taatacgact cactataggg tggtcccaat tctcgtggaa c 41
<210> 9
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
taatacgact cactataggg cttgaagttg accttgatgc c 41

Claims (7)

  1. The application of the LOC663462 gene is characterized in that the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1; the application comprises the following steps: regulating diapause of insects, preparing products for regulating diapause of insects, preventing and controlling pests or preparing products for preventing and controlling pests.
  2. 2. The use according to claim 1, wherein the pest is a coleopteran insect.
  3. The application of the LOC663462 gene as an inhibition target or a silencing target for preparing an interference molecule which specifically interferes with the expression of the harmful insect gene or inhibits the growth of the harmful insect, and is characterized in that the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1.
  4. 4. The use of claim 3, wherein the interfering molecule is a construct of dsRNA, antisense nucleic acid, small interfering RNA, microRNA or capable of expressing or forming said dsRNA, antisense nucleic acid, small interfering RNA, microRNA of LOC663462 gene or its transcript to a target for inhibition or silencing.
  5. 5. The use of claim 4, wherein the primer sequences for synthesizing said dsRNA are shown in SEQ ID NO 2 and SEQ ID NO 3.
  6. 6. A method of controlling harmful insects, said method comprising: down-regulating the expression or activity of LOC663462 gene in the body of the harmful insect; wherein, the nucleotide sequence of the LOC663462 gene is shown as SEQ ID NO. 1.
  7. 7. The method of claim 6, wherein the method of down-regulating the expression or activity of the LOC663462 gene in the insect pest comprises: knocking out or silencing LOC663462 gene in genome of harmful insect; or transferring a down regulator for down-regulating LOC663462 gene transcription, polypeptide expression or polypeptide activity into a harmful insect body.
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Publication number Priority date Publication date Assignee Title
CN110184274B (en) * 2019-05-30 2022-05-27 山西大学 E75 gene and application of dsRNA thereof in pest control
CN112048506B (en) * 2020-08-20 2021-05-11 华南师范大学 dsRNA of BmKRP gene and application thereof in pest control
CN112062824B (en) * 2020-08-20 2021-05-11 华南师范大学 Application of KRP gene in pest control

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PREDICTED: Tribolium castaneum PR domain zinc finger protein 10 (LOC663462), transcript variant X2, mRNA;GenBank;《GenBank》;20160321;第1-2页 *

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